Microbiologists have used the spectrophotometer to measure the specific growth rate of pure cultures of microbes (e.g., bacteria) for over fifty years. For mixed cultures, such as samples collected from the environment and biological reactor systems used by environmental engineers, the spectrophotometer cannot be used to measure the specific growth rate of specific microbial populations. However, several molecular biology-based methods have been described that identify or enumerate distinct microbial populations in mixed culture samples.
The identification and quantification of the abundance of phylogenetically-defined bacterial populations in the environment often is determined using molecular tools targeting 16S rRNA, one of three rRNA molecules in the ribosome. Previously, Oerther and co-workers demonstrated that by targeting the 3′ tail of precursor 16S rRNA, the expression and processing of ribosomal RNA could be monitored with fluorescence in situ hybridizations as an indicator of the physiologic state of Acinetobacter spp. (Oerther, D. B., et al., Appl. Environ. Microbiol., 2000, 66:2154-2165). U.S. Pat. Nos. 5,770,373; 5,726,021; and 5,712,095 describe methods for identifying chloramphenicol resistant strains of mycobacteria. They describe the typical response of ribosome synthesis to chloramphenicol, but do not describe the use of chloramphenicol or other protein synthesis inhibitors to measure the specific rate of ribosome synthesis.
The growth response of individual cells of bacteria can be determined by measuring the level of pre16S rRNA in individual cells (Stroot, P. G. and Oerther, D. B. Water Sci. Technol., 2003, 47(11):241-250). This measurement of pre16S rRNA in individual cells is possible by using FISH with a probe that targets the 3′ end of pre16S rRNA. A new molecular biology method was developed that determines the growth state of an entire bacterial population by determining the level of pre16S rRNA relative to the mature 16S rRNA. Previously, it was demonstrated that the level of pre16S in individual cells of A. calcoaceticus is an indication of their growth state (Stroot, P. G. and Oerther, D. B. Water Sci. Technol., 2003, 47(11):241-250). However, this approach was laborious for mixed cultures, since it required a unique pre probe for each specific bacteria of interest. In addition, the pre16S sequence information available to researchers from the Ribosomal Intergenic Spacer Sequence Collection (RISSC), was restricted to the 3′ precursor region and the total number of sequences was significantly less than the available 16S rRNA sequence information (Garcia-Martinez, J. et al. Nucleic Acids Res., 2001, 29(1):178-80). Cangelosi and Brabant (Cangelosi, G. A. and Brabant, W. H., J. of Bacteriol., 1997, 179(14):4457-4463) used a reverse transcription method to measure the level of precursor 16S rRNA in cells of E. coli that were exposed to chloramphenicol.
It would be advantageous to have available a method that would allow for rapid determination of the specific growth rate of particular microbial populations in a mixed culture.